1.1 Field of the Invention
The present invention is generally directed to the field of nutritional medicine. The present invention is directed to an immunosupportive, drug sparing diet and methods of treating patients employing a nucleotide-free diet. The invention is also related to diet-induced alteration of P450 metabolism via a unique diet that has the potential to restore immunity in patients and decrease pharmaceutical costs associated with treatment. More specifically, the present invention is directed to the use of a nucleotide-free diet that can enhance the immunosuppressive potency of a subtherapeutic dose of drugs metabolized by gut P450 isozymes, such as cyclosporine.
1.2 Description of Related Art
1.2.1 Dietary RNA and the Immune Response
Dietary ribonucleic acid (RNA) is required both for maintenance of normal immune responsiveness and for restoration of lost immune response due to protein deprivation (Van Buren et al., 1990). One of the principal targets is the T-lymphocyte, whose maturation and production of viral cytokines is delayed or suppressed by restriction of dietary nucleotides (Van Buren et al., 1985). Response to pathogens requiring normal T-cell function is suppressed by a nucleotide free diet (NFD). RNA or dietary pyrimidines (uracil) can correct this deficient response while dietary purines (adenine) fail to restore immune responsiveness. Specific immune responses as well as nonspecific immune responses can be influenced by dietary nucleotides (Kulkarni et al., 1986). An LD 50 dose of intravenously injected Staphylococcus aureus is uniformly lethal in mice on a nucleotide free diet. Adenine supplemented diets do not restore host defenses to this bacterial pathogen while a pyrimidine (uracil) supplemented diet is comparable to an RNA supplemented diet in maintaining host immunity against this bacterium. Uniquely, RNA can maintain normal immune responsiveness even during protein starvation and negative nitrogen balance (Pizzi et al., 1990).
Dietary nucleotides are additive to omega-3 fatty acids and arginine in maintaining immune responses to antigenic challenge. These observations resulted in the development of a commercial diet, which in several randomized studies has reduced length of patient hospital stay by over 20% (Bower et al., 1995).
1.2.2 Drug Bioavailability
A pharmaceutical compound (drug), when ingested orally, is absorbed through the various mucosal surfaces, distributed to various tissues through the blood, inactivated by the liver and other tissues, active at the target site, and eliminated in the urine or bile. Factors that affect steps involved with these processes, among others, determine the bioavailability of the drug. For example, if a drug is not efficiently absorbed by the digestive system, this will decrease the percentage of the oral dose that will reach the target tissue. Similarly, if a percentage of the drug is metabolized before it can act on the target tissue, this also decreases the bioavailability of the drug. To compensate for factors that decrease the bioavailability of drugs, higher oral doses are required to elicit the desired effect.
Traditional approaches to increasing the bioavailability of drugs have focused on increasing the solubility of drugs and mucosal membrane permeability. Approaches that have targeted drug metabolism have generally focused on affecting biotransformation in the liver. These methods are inadequate because they affect general liver metabolism and often produce nonspecific systemic effects.
More recently, approaches for increasing bioavailability have targeted drug metabolism in the gut. The cytochrome P450 is responsible for a majority of the biotransformation of drugs in the gut. U.S. Pat. No. 5,716,928 discloses a method of screening compounds that inhibit cytochrome P450 drug metabolism, particularly cytochrome P450 3A in the gut. Through this method, a number of essential oils were shown to inhibit cytochrome P450 activity and thereby potentially increase the bioavailability of drugs coadministered with the oils.
1.2.3 Deficiencies in the Related Art
Dietary RNA is required to maintain or restore the immune system, particularly T-cell function. As RNA is made up of four subunits (adenine, cytosine, guanine, and uracil) and susceptible to degradation, perhaps certain subunits are responsible for the immunosupportive role of dietary RNA while others are not. Indeed, the inventors have determined that pyrimidines, particularly uracil, are responsible for the maintenance and restoration of immune responsiveness (Van Buren et al., 1994).
The immunosuppressive effect of an NFD has been utilized in situations in which an immune response is not wanted. For example, the inventors have shown that mice fed NFD had improved cardiac allograft survival (Van Buren et al., 1983b). Interestingly, in the same study, the inventors found that the NFD and cyclosporine had a synergistic effect. A similar effect was observed by Yau et al. (1991) in their study of the effect of different doses of immunosuppressive drugs on acute graft-vs.-host disease prophylaxis. This group found that drugs given to patients on an NFD had increased efficacy and decreased toxicity. Although NFDs are effective at increasing the efficacy of drugs for purposes of immunosuppression, the lack of nucleotides makes the diets inappropriate in circumstances in which maintaining immune responsiveness is desired. Thus, there is a need for a diet that is immune supportive and drug sparing, that is able to alter drug metabolism to increase the effectiveness/availability of the drug.
It is, therefore, a goal of the present invention to provide an immunosupportive, drug sparing diet. The present invention discloses an immunosupportive, drug sparing diet and methods of treating patients employing an immunosupportive, drug sparing diet. Surprisingly and unexpectedly, the inventors have found that the use of a nucleotide-free diet can synergistically enhance the immunosuppressive potency of a subtherapeutic dose of drugs metabolized by gut P450 isozymes, such as cyclosporine.
Specifically, the inventors have demonstrated that a nucleotide-free diet supplemented with uracil (NFU), or other pyrimidines, fails to stimulate gut P450 enzyme levels and leads to an increase in the bioavailability of drugs metabolized by this enzyme. However, a nucleotide free diet supplemented with adenine (NFA) stimulated expression of P450 in the gut. Therefore, the diets of the present invention are immunosupportive, because they contain compounds (i.e., uracil) that maintain the immune system, yet are drug sparing because they lack compounds (i.e., adenine) that stimulate expression of P450 in the gut. As a result, one can use a diet of the present invention to maintain immune responsiveness and obtain a synergistic potentiation of the activity of drugs metabolized by gut P450. By decreasing drug metabolism, one can reduce pharmacy costs. Additionally, the invented diet at the same time will enhance immune response by the addition of a critical nucleobase.
The present invention encompasses an immunosupportive, drug sparing diet. Another name for diet is a nutritional composition. As used herein, the phrase xe2x80x9cdrug sparingxe2x80x9d refers to the ability of a diet to enhance the bioavailability of an orally administered drug. The drug sparing ability of the diet disclosed herein has been documented by the finding of the inventors of the virtual absence of the p450 enzyme in the gut of mice fed this diet.
To be drug sparing, a diet of the present invention should lack compounds that stimulate the expression of a P450 isozyme in the intestinal tract of an animal receiving the diet. From here on, xe2x80x9cP450 expressionxe2x80x9d or xe2x80x9cexpression of P450xe2x80x9d refers to the expression of P450 isozyme in the intestinal tract of an animal. Disclosed herein are methods of identifying compounds that stimulate the expression of P450. Although in preferred embodiments a diet of the present invention is devoid of any compound that substantially stimulates the expression of P450, a diet of the present invention may contain a relatively small amount of a compound that is found to stimulate P450 expression. However, to be useful for increasing the bioavailability of a drug coadministered with the diet, a diet of the present invention should be functionally pure of any compound that substantially stimulates P450 expression. As used herein xe2x80x9cfunctionally purexe2x80x9d means the level of the compound in the diet is such that the diet remains drug sparing to a useful level.
The inventors have shown that purines and purine analogs increase the expression of P450. In preferred embodiments, the diets of the present invention do not contain, or are substantially free of, those purines and purine analogs that stimulate P450 expression or compounds that may be metabolized into such purines or purine analogs. The purines or purine analogs may be nucleosides or nucleotides and may be present as singular bases or as part of a polynucleotide (i.e., RNA). Purines and purine analogs include adenine, guanine, 2xe2x80x2-O-methylguanosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylguanosine, 2-methylthio-N6-isopentenyladenosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, N6-adenosine, 2,6-diaminopurine, inosine, and 1-methylinosine. In preferred embodiments, the diets of the present invention are lacking adenine. The inventors contemplate that an immunosupportive, drug sparing diet may contain purines or purine analogs and remain drug sparing. However, the level of purines or purine analogs that stimulate P450 expression should be at a concentration such that P450 expression in the gut is not substantially increased. Methods of determining the increase of expression of P450 in the gut are discussed herein.
The term xe2x80x9cimmunosupportivexe2x80x9d refers to the ability of a compound or composition to maintain or restore the immune system in an animal that is administered the compound or composition. Components of the immune system that may be considered when determining immunosupportive properties of diets include cytoxic and helper T-cell functions, macrophage function, or the maintenance and differentiation of stem cells within the recipient of the diet. The inventors have determined that diets comprising pyrimidines, pyrimidine analogs, or compounds that may be metabolized into pyrimidines or pyrimidine analogs are immunosupportive. Pyrimidines and pyrimidine analogs include cytosine, thymine, uracil, 4-acetylcytidine, 5-(carboxyhydroxylmethyl) uridine, 2xe2x80x2-O-methylcytidine, 5-carboxymethylaminomethyl-2-thioridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2xe2x80x2-O-methylpseudouridine, 1-methylpseudouridine, 5-methoxyaminomethyl-2-thiouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, pseudouridine, 2-thiocytidine, 3-methylcytidine, 5-methylcytidine, 5-methylaminomethyluridine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, 2xe2x80x2-O-methyl-5-methyluridine, 2xe2x80x2-O-methyluridine, and 3-(3-amino-3-carboxypropyl)uridine. In preferred embodiments, the diets of the present invention contain uracil. The concentration of uracil in the diet may be from about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, or 0.0006% to about 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, or 6.0%. In more preferred embodiments, uracil comprises from about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, or 0.006% to about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 0.6% of the diet. In yet more preferred embodiments, uracil comprises about 0.06% of the diet.
An important aspect of the nutritional compositions of the present invention are that they do not significantly stimulate the expression of P450 in the intestine of an animal receiving the compositions to a level where P450 enzymes are activated to the point where drugs are metabolized to a level that hinders drug performance. In preferred embodiments, the animal receiving the compositions is a mammal. In the more preferred embodiments, the mammal is a human.
The intestines of an animal are also referred to as the gut. As the gut is made up of the small intestine (duodenum, ileum, and the jejunum), the large intestine (ascending, transversing, and descending), and the colon. The inventors contemplate that the expression of P450 in the small intestine, particularly the jejunum, is responsible for a large portion of drug metabolism in the gut. Of course, since P450 comprises a family of enzymes, the expression of a number of enzymes may affect drug metabolism. However, the inventors contemplate that the CYP3A enzymes are responsible for a large percentage of the drug metabolism in the small intestine.
In an exemplary embodiment, the immunosupportive, drug sparing diet is a nucleotide free diet supplemented with a pyrimidine. In preferred embodiments the pyrimidine is uracil. Generally, a nucleotide free diet comprises 1% to 20% calories from protein, 3% to 25% calories from fat, and the remainder of the calories are provided by carbohydrates. Of course, nucleotide free diets often contain essential vitamins, minerals, and other required defined components.
The present invention also includes methods for increasing the bioavailability of a drug. These methods comprise administering a diet of the present invention to an animal in need of treatment of the drug. Because the diets of the present invention do not significantly stimulate the expression of P450 relative to a nucleotide free diet, the amount of drug that is metabolized by the gut is decreased thereby increasing the bioavailability of the drug. In preferred embodiments, the drug is sirolimus, sildenafil, tacrolimus, erythromycin, zithromycin, or cyclosporine. In an exemplary embodiment, the drug is cyclosporine. However, the inventors contemplate that essentially any drug that is metabolized by P450 will have increased bioavailability when coadministered with a diet of the present invention.
Although the administration of a drug and a diet of the present invention may be initiated concurrently. In preferred embodiments, administration of a diet of the present invention is initiated about three weeks prior to administration of the drug and continued to be administered concurrently with the drug.
Also provided by the present invention are methods of decreasing P450 metabolism. As used herein, xe2x80x9cP450 metabolismxe2x80x9d refers to the ability of the P450 isozymes in the intestine to metabolize a substrate. The methods of decreasing P450 metabolism comprise administering to an animal an immunosupportive diet lacking a compound that causes substantial stimulation of expression of a P450 isozyme in the intestinal tract.
Further provided are methods of identifying a compound that increases the expression of a P450 isozyme in the intestine of an animal. The methods comprise providing a nucleotide free diet containing a compound suspected of increasing the expression of P450 to an animal. Of course, a range of concentrations of the compound may be compared to determine if the effect of the compound of P450 expression is dose dependent. To determine the effect of the compound on P450 expression, extracts are made from the intestine of the animals receiving the compound. In preferred embodiments, the extracts are made from the small intestine. The extracts may comprise protein or RNA. Level of expression is determined by measuring the amount of P450 isozyme mRNA present in an RNA extract or P450 isozyme protein in a protein extract. In preferred embodiments, the isozyme is CYP3A. In the most preferred embodiment, the isozyme is CYP3A2. Methods of determining the amount of a specific mRNA or protein in an extract is provided herein and is well known to those of skill in the art.
To determine the effect of the compound on P450 expression, one may compare the level of expression of P450 in an animal administered a nucleotide free diet comprising the compound to that of an animal administered the nucleotide diet without the compound. A higher level of expression of P450 in an animal administered the nucleotide free diet comprising the compound than the level of expression of P450 in an animal administered the nucleotide free diet without the compound is indicative that the compound is able to stimulate P450 expression.
The inventors contemplate that one may use the above method of identifying compounds that increase the expression of P450 to select compounds that may be used in conjunction with the diets of the present invention. If a compound or concentration of a compound is determined to stimulate expression of P450, then that compound or concentration of the compound may be used in methods in which one wishes to increase the drug metabolism in a patient (i.e., to treat drug overdose). However, if a compound or concentration of compound is found not to stimulate expression of P450 using this method, then that compound or concentration of compound may be used in the drug sparing diets of the present invention.
As used in the specification and claims, following long-standing patent law practice, the terms xe2x80x9caxe2x80x9d and xe2x80x9can,xe2x80x9d when used in conjunction with the word xe2x80x9ccomprising means one or more.